Erika B. Villanueva

Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS.

Autosomal dominant diseases such as Huntington’s disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with ∼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs—an outcome with broad application to HD and other dominant genetic disorders.

In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides.

Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD.

A fully humanized transgenic mouse model of Huntington disease

Silencing the mutant huntingtin gene (muHTT) is a direct and simple therapeutic strategy for the treatment of Huntington disease (HD) in principle. However, targeting the HD mutation presents challenges because it is an expansion of a common genetic element (a CAG tract) that is found throughout the genome. Moreover, the HTT protein is important for neuronal health throughout life, and silencing strategies that also reduce the wild-type HTT allele may not be well tolerated during the long-term treatment of HD. Several HTT silencing strategies are in development that target genetic sites in HTT that are outside of the CAG expansion, including HD mutation-linked single-nucleotide polymorphisms and the HTT promoter. Preclinical testing of these genetic therapies has required the development of a new mouse model of HD that carries these human-specific genetic targets. To generate a fully humanized mouse model of HD, we have cross-bred BACHD and YAC18 on the Hdh(-/-) background. The resulting line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD having two human HTT genes, no mouse Hdh genes and heterozygosity of the HD mutation. We find that Hu97/18 mice display many of the behavioral changes associated with HD including motor, psychiatric and cognitive deficits, as well as canonical neuropathological abnormalities. This mouse line will be useful for gaining additional insights into the disease mechanisms of HD as well as for testing genetic therapies targeting human HTT.

Synthesis and biological evaluation of novel pyrazolyl-2,4-thiazolidinediones as anti-inflammatory and neuroprotective agents

Novel pyrazolyl-2,4-thiazolidinediones were prepared via the reaction of appropriate pyrazolecarboxaldehydes with 2,4-thiazolidinediones and substituted benzyl-2,4-thiazolidinediones. The resultant compounds were first evaluated for their anti-inflammatory and neuroprotective properties in vitro. The active compounds were further studied in vivo by using the formalin-induced paw edema and the turpentine oil-induced granuloma pouch bioassays. We identified four novel compounds that showed protective effects in vitro at non-toxic concentrations, and were also effective in the animal models of acute and sub-acute inflammation.

Anti-semaphorin 4D immunotherapy ameliorates neuropathology and some cognitive impairment in the YAC128 mouse model of Huntington disease

Huntington disease (HD) is an inherited, fatal neurodegenerative disease with no disease-modifying therapy currently available. In addition to characteristic motor deficits and atrophy of the caudate nucleus, signature hallmarks of HD include behavioral abnormalities, immune activation, and cortical and white matter loss. The identification and validation of novel therapeutic targets that contribute to these degenerative cellular processes may lead to new interventions that slow or even halt the course of this insidious disease. Semaphorin 4D (SEMA4D) is a transmembrane signaling molecule that modulates a variety of processes central to neuroinflammation and neurodegeneration including glial cell activation, neuronal growth cone collapse and apoptosis of neural precursors, as well as inhibition of oligodendrocyte migration, differentiation and process formation. Therefore, inhibition of SEMA4D signaling could reduce CNS inflammation, increase neuronal outgrowth and enhance oligodendrocyte maturation, which may be of therapeutic benefit in the treatment of several neurodegenerative diseases, including HD. To that end, we evaluated the preclinical therapeutic efficacy of an anti-SEMA4D monoclonal antibody, which prevents the interaction between SEMA4D and its receptors, in the YAC128 transgenic HD mouse model. Anti-SEMA4D treatment ameliorated neuropathological signatures, including striatal atrophy, cortical atrophy, and corpus callosum atrophy and prevented testicular degeneration in YAC128 mice. In parallel, a subset of behavioral symptoms was improved in anti-SEMA4D treated YAC128 mice, including reduced anxiety-like behavior and rescue of cognitive deficits. There was, however, no discernible effect on motor deficits. The preservation of brain gray and white matter and improvement in behavioral measures in YAC128 mice treated with anti-SEMA4D suggest that this approach could represent a viable therapeutic strategy for the treatment of HD. Importantly, this work provides in vivo demonstration that inhibition of pathways initiated by SEMA4D constitutes a novel approach to moderation of neurodegeneration.

Ultrasensitive measurement of huntingtin protein in cerebrospinal fluid demonstrates increase with Huntington disease stage and decrease following brain huntingtin suppression

Quantitation of huntingtin protein in the brain is needed, both as a marker of Huntington disease (HD) progression and for use in clinical gene silencing trials. Measurement of huntingtin in cerebrospinal fluid could be a biomarker of brain huntingtin, but traditional protein quantitation methods have failed to detect huntingtin in cerebrospinal fluid. Using micro-bead based immunoprecipitation and flow cytometry (IP-FCM), we have developed a highly sensitive mutant huntingtin detection assay. The sensitivity of huntingtin IP-FCM enables accurate detection of mutant huntingtin protein in the cerebrospinal fluid of HD patients and model mice, demonstrating that mutant huntingtin levels in cerebrospinal fluid reflect brain levels, increasing with disease stage and decreasing following brain huntingtin suppression. This technique has potential applications as a research tool and as a clinical biomarker.

Mitochondrial transcription factor A (Tfam) is a pro-inflammatory extracellular signaling molecule recognized by brain microglia.

Microglia represent mononuclear phagocytes in the brain and perform immune surveillance, recognizing a number of signaling molecules released from surrounding cells in both healthy and pathological situations. The microglia interact with several damage-associated molecular pattern molecules (DAMPs) and recent data indicate that mitochondrial transcription factor A (Tfam) could act as a specific DAMP in peripheral tissues. This study tested the hypothesis that extracellular Tfam induces pro-inflammatory and cytotoxic responses of the microglia. Three different types of human mononuclear phagocytes were used to model human microglia: human peripheral blood monocytes from healthy donors, human THP-1 monocytic cells, and human primary microglia obtained from autopsy samples. When combined with interferon (IFN)-γ, recombinant human Tfam (rhTfam) induced secretions that were toxic to human SH-SY5Y neuroblastoma cells in all three models. Similar cytotoxic responses were observed when THP-1 cells and human microglia were exposed to human mitochondrial proteins in the presence of IFN-γ. rhTfam alone induced expression of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and IL-8 by THP-1 cells. This induction was further enhanced in the presence of IFN-γ. Upregulated secretion of IL-6 in response to rhTfam plus IFN-γ was confirmed in primary human microglia. Use of specific inhibitors showed that the rhTfam-induced cytotoxicity of human THP-1 cells depended partially on activation of c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (MAPK). Overall, our data support the hypothesis that, in the human brain, Tfam could act as an intercellular signaling molecule that is recognized by the microglia to cause pro-inflammatory and cytotoxic responses.

Synthesis and biological evaluation of novel pyrazole compounds

A novel dipyrazole ethandiamide compound and acid chloride of pyrazolo[3,4-d]pyrimidine 4(5H)-one were prepared and reacted with a number of nucleophiles. The resultant novel compounds were tested in several in vitro and in vivo assays. Three compounds inhibited the secretion of neurotoxins by human THP-1 monocytic cells at concentrations that were not toxic to these cells. They also partially inhibited both cyclooxygenase-1 and -2 isoforms. In animal studies, two compounds were notable for their anti-inflammatory activity that was comparable to that of the clinically available cyclooxygenase-2 inhibitor celecoxib. Modeling studies by using the molecular operating environment module showed comparable docking scores for the two enantiomers docked in the active site of cyclooxygenase-2.

An enhanced Q175 knock-in mouse model of Huntington disease with higher mutant huntingtin levels and accelerated disease phenotypes

Huntington disease (HD) model mice with heterozygous knock-in (KI) of an expanded CAG tract in exon 1 of the mouse huntingtin (Htt) gene homolog genetically recapitulate the mutation that causes HD, and might be favoured for preclinical studies. However, historically these mice have failed to phenotypically recapitulate the human disease. Thus, homozygous KI mice, which lack wildtype Htt, and are much less relevant to human HD, have been used. The zQ175 model was the first KI mouse to exhibit significant HD-like phenotypes when heterozygous. In an effort to exacerbate HD-like phenotypes and enhance preclinical utility, we have backcrossed zQ175 mice to FVB/N, a strain highly susceptible to neurodegeneration. These Q175F mice display significant HD-like phenotypes along with sudden early death from fatal seizures. The zQ175 KI allele retains a floxed neomycin resistance cassette upstream of the Htt gene locus and produces dramatically reduced mutant Htt as compared to the endogenous wildtype Htt allele. By intercrossing with mice expressing cre in germ line cells, we have excised the neo cassette from Q175F mice generating a new line, Q175FΔneo (Q175FDN). Removal of the neo cassette resulted in a ∼2 fold increase in mutant Htt and rescue of fatal seizures, indicating that the early death phenotype of Q175F mice is caused by Htt deficiency rather than by mutant Htt. Additionally, Q175FDN mice exhibit earlier onset and a greater variety and severity of HD-like phenotypes than Q175F mice or any previously reported KI HD mouse model, making them valuable for preclinical studies.

Secreted phospholipase A2 group IIA is a neurotoxin released by stimulated human glial cells

Neuroinflammation, which is one of the hallmarks of neurodegenerative disorders such as Alzheimers disease, involves secretion of pro-inflammatory mediators by activated glial cells. Secreted phospholipase A(2) group IIA (sPLA(2)IIA) has been implicated as an inflammatory mediator contributing to various peripheral inflammatory conditions; however, little is known about the role this enzyme plays in neuroinflammation. Human microglia-like promonocytic THP-1 cells and human primary astrocytes were used to study sPLA(2)IIA expression, secretion and function. Production of sPLA(2)IIA by these cells was induced in response to stimulation by pro-inflammatory mediators at both mRNA and protein levels. Removal of sPLA(2)IIA from stimulated human microglia-like cell and astrocyte supernatants by immunosorbent caused significant reduction of their toxicity towards SH-SY5Y neuroblastoma cells. Both sPLA(2)IIA specific and non-specific PLA(2) inhibitors exhibited no anti-cytotoxic or neuroprotective effects, suggesting that sPLA(2)IIA cytotoxicity is mediated by a non-enzymatic mechanism. The data obtained indicate that sPLA(2)IIA may contribute to the pathogenesis of neurodegenerative diseases involving neuroinflammation. Agents inhibiting the non-enzymatic actions of sPLA(2)IIA could be used to slow down progression of neurodegenerative processes that are driven by inflammation.